Project Objective This web page was created as part of an assignment in Emory University's Biology 142 lab course. Students were assigned proteins to be researched, in order to learn about the function and structure of each protein, and determine whether the newly sequenced whale shark genome had evidence of the same proteins. This page is an analysis PSMB8.
Background PSMB8 (Proteasome subunit beta type-8) plays an unique role in controlling pathogenic immune responses. Figure 1 below shows the general function of an immunoproteasome. The immunoproteasome, a modified proteasome, is found predominantly in white blood cells. It shapes the antigenic repertoire presented on MHC-I, which is a molecule to display fragments of proteins from within the cell to the white blood cells (Schmidt et al 1999). PSMB8 is selectively inhibited by a subunit of the immunoproteasome called PR-957. PR-957 blocks presentation of PSMB8-specific antigens either in a whole, living organism, or in a controlled environment outside of a living organism (Kitamura et al 2011).
PSMB8 is also critically involved in the differentiation of fat cells from preadipocytes into adipocytes. Proteasomes helps maintain cellular function through the selective degradation of modified proteins (Kitamura et al 2011). A homozygous missense mutation in PSMB8 increases assembly intermediates of immunoproteasomes. This mutation results in a decreased function of proteasome and a accumulation of modified protein in the patient’s tissues. It indicates that immunoproteasomes have multiple functions in maintaining the homeostasis of a variety of cell types (Agarwal et al 2010).
Figure 1. Alpha and Beta-type cells make up the structure of the immunoproteasome, with alpha being the purple region at the top, and each brown circular region represents one beta-type, such as beta-type 8 in the PSMB8 protein. The immunoproteasome takes in pathogens and various other substances, and performs various tasks such as regulation of cytokine production (Frontiers in Bioscience). Methods
Whale Shark Orthologs
The Galaxy server (whaleshark.georgiaaquarium.org) was utilized to run a BLAST against the predicted whale shark protein database and the human protein sequence (ENSP00000406878) was used as the query. The top five predicted protein hits were identified and the FASTA DNA sequences were extracted. The full predicted whale shark sequences, not just the aligned portion, were used as queries to BLAST against the NCBI human protein database (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Orthologs
PSMB8 predicted orthologs were identified in species other than whale sharks using the NCBI Blast server. Protein BLASTs were performed using single species protein databases for mouse, zebra fish and elephant shark protein databases. The human PSMB8 protein (ENSP00000406878) was used as query sequence in these searches with default settings.
Phylogenetic Tree
The human sequence was the query from which the other species' sequences were compare to. The top 5 hits of the whale shark predicted protein along with zebrafish, elephant shark, and mouse BLAST hits were used to create the phylogenetic tree, specifically using ClustalW2 to create a multiple sequence alignment.
Results
PSMB8 in the Whale Shark The results of the BLAST performed in the Galaxy server of the whale shark database with the human protein sequence as the query are shown below in Table 1. Five hits each with an e-value below 3e-08 were identified. The best predicted hit had an e-value of 1e-101
Whale Shark ID
e-value
Alignment Length
Predicted Protein Length
% Identity
g21003.t1
1e-101
194
276
66.49
g28391.t1
7e-39
111
276
57.66
g26495.t1
3e-36
68
276
79.41
g36697.t1
4e-14
125
276
28.00
g26434.t1
3e-08
65
276
32.31
Table 1. Human PSMB8 top Blastp hits from whale shark predicted protein database. This was done using the Galaxy server from the Georgia Aquarium using the human protein sequence as the query. This table displays whale shark ID, alignment length, and % identity of the top 5 hits according to lowest e-values generated. The predicted whale shark protein with an ID of g21003.t1 returned PSMB8 as the best hit against the NCBI human protein database. The process was repeated using the elephant shark, mouse, and zebrafish predicted PSMB8 proteins to identify protein domains, identify more orthologs, and build a phylogenetic tree.
Protein domains: The best hit whale shark protein sequence with an ID of g21003.t1is aligning with the entire sequence of the human PSMB8 protein sequence (from BLAST results). This indicates that the best hit whale shark PSMB8 protein sequence and the human PSMB8 protein sequence are homologous sequences. Potential PSMB8-like proteins in the whale shark (from BLAST results) all contain both a proteasome subunit domain and a Ntn_hydrolase superfamily (Figure 2). The proteasome subunit domains are involved in the degradation of unneeded or damaged proteins by breaking the peptide bonds (Valas and Bourne 2008). Ntn_hydrolase superfamily is a diverse superfamily of of enzymes that are activated autocatalytically. All known members catalyze the hydrolysis of amide bonds in either proteins or small molecules, and each one of them is synthesized as a preprotein (Oinonen and Rouvinen).
top hit.png
Figure 2: Putative domains of whale shark PSMB8 best hit predicted proteins. All of the five best-hit whale shark predicted proteins contain putative proteasome subunit domain and a Ntn_hydrolase superfamily as predicted by NCBI BLAST server analyses. Orthologs: The whale shark PSMB8 protein sequence (ENSP00000406878) was used as query in the NCBI BLAST searches against the protein databases of mice, zebrafish, yeast, fruit fly and elephant sharks. The best hits for each species were listed in Table 2. PSMB8 orthologues were found in mice, zebrafish and elephant shark. Yeast and fruit fly all contain proteasome domain proteins but not necessarily PSMB8 orthologues.
proteasome beta5 subunit, isoform A [Drosophila melanogaster]
NP_652014.1
282
84%
2.00E-92
56%
Table 2. The whale shark PSMB8 sequence was used in protein BLASTs against individual species. Name, ID, length, Query coverage, E-value and Identity of the best hit from each search is reported here.
Phylogeny:
Two of the top whale shark hits (g26434.t1 and g36697.t1) are the oldest in relation to the other sequences, and closest in relation to the whale shark sequence, while the sequence of g28391.t1 is close in relation to the elephant shark, which are both slightly younger evolutionarily speaking. Logically, the zebrafish sequence is further along on the tree, as it evidently evolved later on. The mouse and human sequences, which are closely related as both species are mammals, occupy the opposite end of the tree from the g26434.t1 sequence, as they are the youngest.
psmb8.png
Figure 3. The top five BLAST hits along with other species' sequences were used in ClustalW2 to create a phylogenetic tree.
Conclusion We were able to identify predicted PSMB8 orthologs in whale sharks. This was done by using the Blast software to compare the human protein sequence to the predicted whale shark protein sequence. The best five whale shark ID sequences were identified to be g21003.t1, g28391.t1, g36697.t1, g26434.t1, and g26495.t1. More orthologs were found using the NCBI database using the human protein sequence as the query for the mouse, zebrafish, and elephant shark databases. Finally, these sequences were used to construct a phylogenetic tree that depicted evolution of the protein. The whale shark protein tags g28391.t1 and g21003.t1 were found to be most closely related to the elephant shark, which has previously been identified as the whale shark's closest known relative. More research need to be done to find specifically where the gene that codes for the PSMB8 protein falls in the overall whale shark genome. This is important because of PSMB8's critical role in controlling pathogenic immune response and differentiation of fat cells.
References:
Kitamura A., Maekawa Y., Uehara H., Izumi K. , Izumi K., Nishizawa M., Toyoshima Y., Takahashi H., Standley D.M., Tanaka K., Hamazaki J., Murata S., Obara K., Toyoshima I., and Yasutomo K. (2011). A mutation in the immunoproteasome subunit PSMB8 causes autoinflammation and lipodystrophy in humans. Journal of Clinical Investigation 121(10):4150-4160.
Schmidt M, Zantopf D, Kraft R, Kostka S, Preissner R, Kloetzel PM (Jun 1999). "Sequence information within proteasomal prosequences mediates efficient integration of beta-subunits into the 20 S proteasome complex". J Mol Biol 288 (1): 117–28. doi:10.1006/jmbi.1999.2660. PMID10329130.
Tony Muchamuel, Michael Basler, Monette A Aujay, Erika Suzuki, Khalid W Kalim, Christoph Lauer, Catherine Sylvain, Eileen R Ring, Jamie Shields, Jing Jiang, Peter Shwonek, Francesco Parlati, Susan D Demo, Mark K Bennett, Christopher J Kirk & Marcus Groettrup (June 2009). “A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis”. Nature Medicine 15, 781 - 787
Oinonen, C. and Rouvinen, J. (2000). Structural comparison of Ntn-hydrolases. Protein Science : A Publication of the Protein Society, 9(12), 2329–2337.
Agarwal, A. K., Xing, C., DeMartino, G. N., Mizrachi, D., Hernandez, M. D., Sousa, A. B., Martinez de Villarreal, L., dos Santos, H. G., Garg, A. PSMB8 encoding the beta-5i proteasome subunit is mutated in joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced lipodystrophy syndrome. Am. J. Hum. Genet. 87: 866-872, 2010. PubMed: 21129723, related citations
This web page was created as part of an assignment in Emory University's Biology 142 lab course. Students were assigned proteins to be researched, in order to learn about the function and structure of each protein, and determine whether the newly sequenced whale shark genome had evidence of the same proteins. This page is an analysis PSMB8.
Background
PSMB8 (Proteasome subunit beta type-8) plays an unique role in controlling pathogenic immune responses. Figure 1 below shows the general function of an immunoproteasome. The immunoproteasome, a modified proteasome, is found predominantly in white blood cells. It shapes the antigenic repertoire presented on MHC-I, which is a molecule to display fragments of proteins from within the cell to the white blood cells (Schmidt et al 1999). PSMB8 is selectively inhibited by a subunit of the immunoproteasome called PR-957. PR-957 blocks presentation of PSMB8-specific antigens either in a whole, living organism, or in a controlled environment outside of a living organism (Kitamura et al 2011).
PSMB8 is also critically involved in the differentiation of fat cells from preadipocytes into adipocytes. Proteasomes helps maintain cellular function through the selective degradation of modified proteins (Kitamura et al 2011). A homozygous missense mutation in PSMB8 increases assembly intermediates of immunoproteasomes. This mutation results in a decreased function of proteasome and a accumulation of modified protein in the patient’s tissues. It indicates that immunoproteasomes have multiple functions in maintaining the homeostasis of a variety of cell types (Agarwal et al 2010).
Figure 1. Alpha and Beta-type cells make up the structure of the immunoproteasome, with alpha being the purple region at the top, and each brown circular region represents one beta-type, such as beta-type 8 in the PSMB8 protein. The immunoproteasome takes in pathogens and various other substances, and performs various tasks such as regulation of cytokine production (Frontiers in Bioscience).
Methods
Whale Shark Orthologs
The Galaxy server (whaleshark.georgiaaquarium.org) was utilized to run a BLAST against the predicted whale shark protein database and the human protein sequence (ENSP00000406878) was used as the query. The top five predicted protein hits were identified and the FASTA DNA sequences were extracted. The full predicted whale shark sequences, not just the aligned portion, were used as queries to BLAST against the NCBI human protein database (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Orthologs
PSMB8 predicted orthologs were identified in species other than whale sharks using the NCBI Blast server. Protein BLASTs were performed using single species protein databases for mouse, zebra fish and elephant shark protein databases. The human PSMB8 protein (ENSP00000406878) was used as query sequence in these searches with default settings.
Phylogenetic Tree
The human sequence was the query from which the other species' sequences were compare to. The top 5 hits of the whale shark predicted protein along with zebrafish, elephant shark, and mouse BLAST hits were used to create the phylogenetic tree, specifically using ClustalW2 to create a multiple sequence alignment.
Results
PSMB8 in the Whale Shark
The results of the BLAST performed in the Galaxy server of the whale shark database with the human protein sequence as the query are shown below in Table 1. Five hits each with an e-value below 3e-08 were identified. The best predicted hit had an e-value of 1e-101
Table 1. Human PSMB8 top Blastp hits from whale shark predicted protein database. This was done using the Galaxy server from the Georgia Aquarium using the human protein sequence as the query. This table displays whale shark ID, alignment length, and % identity of the top 5 hits according to lowest e-values generated.
The predicted whale shark protein with an ID of g21003.t1 returned PSMB8 as the best hit against the NCBI human protein database. The process was repeated using the elephant shark, mouse, and zebrafish predicted PSMB8 proteins to identify protein domains, identify more orthologs, and build a phylogenetic tree.
Protein domains:
The best hit whale shark protein sequence with an ID of g21003.t1is aligning with the entire sequence of the human PSMB8 protein sequence (from BLAST results). This indicates that the best hit whale shark PSMB8 protein sequence and the human PSMB8 protein sequence are homologous sequences. Potential PSMB8-like proteins in the whale shark (from BLAST results) all contain both a proteasome subunit domain and a Ntn_hydrolase superfamily (Figure 2). The proteasome subunit domains are involved in the degradation of unneeded or damaged proteins by breaking the peptide bonds (Valas and Bourne 2008). Ntn_hydrolase superfamily is a diverse superfamily of of enzymes that are activated autocatalytically. All known members catalyze the hydrolysis of amide bonds in either proteins or small molecules, and each one of them is synthesized as a preprotein (Oinonen and Rouvinen).
Figure 2: Putative domains of whale shark PSMB8 best hit predicted proteins. All of the five best-hit whale shark predicted proteins contain putative proteasome subunit domain and a Ntn_hydrolase superfamily as predicted by NCBI BLAST server analyses.
Orthologs:
The whale shark PSMB8 protein sequence (ENSP00000406878) was used as query in the NCBI BLAST searches against the protein databases of mice, zebrafish, yeast, fruit fly and elephant sharks. The best hits for each species were listed in Table 2. PSMB8 orthologues were found in mice, zebrafish and elephant shark. Yeast and fruit fly all contain proteasome domain proteins but not necessarily PSMB8 orthologues.
Phylogeny:
Two of the top whale shark hits (g26434.t1 and g36697.t1) are the oldest in relation to the other sequences, and closest in relation to the whale shark sequence, while the sequence of g28391.t1 is close in relation to the elephant shark, which are both slightly younger evolutionarily speaking. Logically, the zebrafish sequence is further along on the tree, as it evidently evolved later on. The mouse and human sequences, which are closely related as both species are mammals, occupy the opposite end of the tree from the g26434.t1 sequence, as they are the youngest.
Figure 3. The top five BLAST hits along with other species' sequences were used in ClustalW2 to create a phylogenetic tree.
Conclusion
We were able to identify predicted PSMB8 orthologs in whale sharks. This was done by using the Blast software to compare the human protein sequence to the predicted whale shark protein sequence. The best five whale shark ID sequences were identified to be g21003.t1, g28391.t1, g36697.t1, g26434.t1, and g26495.t1. More orthologs were found using the NCBI database using the human protein sequence as the query for the mouse, zebrafish, and elephant shark databases. Finally, these sequences were used to construct a phylogenetic tree that depicted evolution of the protein. The whale shark protein tags g28391.t1 and g21003.t1 were found to be most closely related to the elephant shark, which has previously been identified as the whale shark's closest known relative. More research need to be done to find specifically where the gene that codes for the PSMB8 protein falls in the overall whale shark genome. This is important because of PSMB8's critical role in controlling pathogenic immune response and differentiation of fat cells.
References: